toxins Article Three-Finger Toxins from Brazilian Coral Snakes: From Molecular Framework to Insights in Biological Function Jessica Matos Kleiz-Ferreira 1,2, Nuria Cirauqui 3, Edson Araujo Trajano 1,2, Marcius da Silva Almeida 2 and Russolina Benedeta Zingali 1,* 1 Laboratório de Hemostase e Venenos—Instituto de Bioquímica Médica, Leopoldo de Meis (IBqM) and Instituto Nacional de Ciência e Tecnologia de Biologia Estrutural e Bioimagem (Inbeb)—Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro 21941-902, Brazil; [email protected] (J.M.K.-F.); [email protected] (E.A.T.) 2 Protein Advanced Biochemistry (PAB), Instituto de Bioquímica Médica Leopoldo de Meis (IBqM) and Centro Nacional de Biologia Estrutural e Bioimagem (CENABIO), Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-902, Brazil; [email protected] 3 Faculdade de Farmacia, Universidade Federal do Rio de Janeiro (UFRJ), Rio de Janeiro 21941-902, Brazil; [email protected] * Correspondence: [email protected] Abstract: Studies on 3FTxs around the world are showing the amazing diversity in these proteins both in structure and function. In Brazil, we have not realized the broad variety of their amino acid sequences and probable diversified structures and targets. In this context, this work aims to conduct an in silico systematic study on available 3FTxs found in Micrurus species from Brazil. We elaborated a specific guideline for this toxin family. First, we grouped them according to their structural homologue predicted by HHPred server and further curated manually. For each group, we selected one sequence and constructed a representative structural model. By looking at conserved features and comparing with the information available in the literature for this toxin family, we Citation: Kleiz-Ferreira, J.M.; managed to point to potential biological functions. In parallel, the phylogenetic relationship was Cirauqui, N.; Trajano, E.A.; Almeida, estimated for our database by maximum likelihood analyses and a phylogenetic tree was constructed M.d.S.; Zingali, R.B. Three-Finger including the homologous 3FTx previously characterized. Our results highlighted an astonishing Toxins from Brazilian Coral Snakes: diversity inside this family of toxins, showing some groups with expected functional similarities to From Molecular Framework to known 3FTxs, and pointing out others with potential novel roles and perhaps structures. Moreover, Insights in Biological Function. Toxins this classification guideline may be useful to aid future studies on these abundant toxins. 2021, 13, 328. https://doi.org/ 10.3390/toxins13050328 Keywords: Micrurus venoms; three-finger toxins; sequence variability; structure-function; classifica- Received: 9 April 2021 tion guideline Accepted: 28 April 2021 Published: 30 April 2021 Key Contribution: Three-finger toxins possess high sequence variability and diversified function, being a prominent pharmacological tool. We developed a new way to cluster toxins with distinguished variability Publisher’s Note: MDPI stays neutral in the main primary structure. We classified 57 three-finger toxin sequences into nine groups, based on with regard to jurisdictional claims in structural features. Toxins that are structurally similar to other 3FTxs already characterized and peculiar published maps and institutional affil- toxins which may present novel functions were identified. This work sheds light on the molecular and iations. biological properties of some 3FTxs from Brazilian Micrurus coral snakes. Copyright: © 2021 by the authors. 1. Introduction Licensee MDPI, Basel, Switzerland. In Brazil, the Micrurus genera of coral snakes represents almost the totality of elapids, This article is an open access article counting about 34 species [1] widespread throughout the country. Just as other coral distributed under the terms and snakes, the venoms of Brazilian Micrurus are predominant in three-finger toxins (3FTxs) conditions of the Creative Commons and phospholipase A2 (PLA2), along with other less abundant proteins [2–4]. They may Attribution (CC BY) license (https:// cause several injuries on envenomed animals such as myotoxicity, edema, nephrotoxicity, creativecommons.org/licenses/by/ hemorrhages, and neurotoxicity. In humans, the major harm inflicted by these venoms is 4.0/). Toxins 2021, 13, 328. https://doi.org/10.3390/toxins13050328 https://www.mdpi.com/journal/toxins Toxins 2021, 13, 328 2 of 19 the blockage of neuromuscular junction by the action of pre and post-synaptic toxins, the PLA2s and the 3FTxs, which may lead to an outcome of respiratory arrest [5,6]. The 3FTxs belong to a family of non-enzymatic proteins constituted by approximately 58 to 90 amino acid residues. In all members of the family characterized to date, the protein fold is based on three loops of β-strands that resemble “fingers” extending to a globu- lar core, stabilized by four conserved disulfide bridges [7]. These toxins present at least eight cysteines commonly well conserved, but also may present nine, ten, or even eleven cysteines [3,7]. This group of toxins is very exquisite and diversified in terms of primary structure and biological roles. This diversification has been proposed to be associated with the theory in which exonic regions in 3FTx genes have been exchanged by others. For in- stance, it can cause a shift in segments that may alter local structures and charge surface (accelerated segment switch in exons to alter targeting—ASSET mechanism) [8] generating the observed sequence diversity bounded to the multi-functionality and diversified targets. In concert, the prey–predator battlefield causes an evolutionary race, where negative and positive selection of key amino acids may also take place [9]. A wide array of biological roles is described for these toxins. One of the classical and well described activities is the agonism and antagonism of cholinergic receptors [10–12]. Updates in the last decade listed other 3FTx functions, including modulation of GABA A receptors [13–15], inhibition of acid-sensing ion channels (ASICs) [16], modulation of adrenoreceptors [17–20], activation of potassium channel [21], activation of voltage-gated sodium channel [22], activation of sperm-mobility [23], induction of insulin secretion from β-cells [24,25], among many others. In the venoms from Brazilian Micrurus species, the amount of 3FTxs is very signifi- cant, even reaching 95% of the whole venom, as in the case of the Micrurus surinamensis species [26]. This represents a vast universe of possibilities of functions to be explored. Even though, few studies have been published to date about these 3FTxs from Brazilian Micrurus species. Considering the characterization of the 3FTxs in Brazilian venoms per se, meaning the identification of their molecular biology and functions, just a few articles were found, and these did not deepen the understanding of their function and molecular mechanism of action [27–29]. The last years of molecular biology and pharmacological studies on 3FTxs around the world in the frame of toxinology show all the diversity in amino acid sequence, structure, and function of these proteins. As mentioned, in Brazil barely anything is known, leaving a gap in the knowledge of these incredible toxins. In this way, this work aimed to conduct a systematic study on 3FTxs from Brazilian Micrurus species, focusing on their amino acid sequences and predicted 3D structures. By doing so, we bring insights on biological function, and also in the phylogenetic relationship of these toxins, which may guide further functional characterizations. 2. Results 2.1. Three-Finger Toxins Classification Based on Protein Threading Three-dimensional protein structure undergoes evolutionary changes more ponder- ously than the protein sequence itself. This knowledge is well exemplified in the 3FTx family. They present a remarkably diversified primary structure, despite the overall three- dimensional conformation of “3 fingers” being conserved in all protein-members of this family. For this reason, we proposed a protein-classification based on a protein-threading algorithm, which takes into account conserved structural patterns (see methods). We used the HHPred software to select the most probable structural homologue for each protein sequence used in the present study, by using an implemented algorithm for hidden Markov models comparison (HMM-HMM) in HHPred. This method allows sensi- tive detection of a structural homologue since it is based on profile-sequence comparison, not just sequence-sequence comparison [30–33]. As a first step, we grouped 57 sequences of our dataset (Table S1) according to the best hit with the lowest E-value suggested by HHPred, curated manually. To further verify and refine this first classification, and to get the final structural homologue, we analyzed Toxins 2021, 13, 328 3 of 19 conserved sequence-patterns, finger extension, and disulfide bridges in those sequences (Table S2). For example, we observed that some sequences have a prolongation of the middle finger, as in the case of group 8. Other examples are groups 2 and 7, in which we found a 5th extra disulfide bond in the first finger. Thus, we verified if sequences with distinguished structure were gathered into groups in line with HHPred results. Afterward, some groups were merged together as subgroups, due to the proximity both in homologous function-structure characteristics and in phylogenetic analysis. Moreover, the sequence length did not seem to be related to group separation according to this method-analysis, as we found a combination of sequences with different lengths grouped together. We ended up with a total of 9 groups, group 1 being subdivided into three subgroups (named 1-A to 1-C), group 2 subdivided into two subgroups (named 2-A and 2-B), and group 8 also subdivided into two subgroups (8-A and 8-B) (Figure1, Figure2 and Table S2). Figure 1. Sequence alignment of 3FTx groups. Groups are presented with their consensus amino acids and conservation score on a scale from 0 to 10 (10 being indicated by an asterisk as completely conserved, and + for similar amino acids). The cysteine residues forming the disulfide bridges are linked by black lines on the top of the alignment.